System and method for absolute quantitation of proteins

ABSTRACT

Absolute quantitation of protein in a sample is provided by comparing a sum or average of the N highest ionization intensities observed for peptides of a particular protein along with a calibration standard. The calibration standard can be in the form of a table generated by prior protein peptide analysis performed using one or more pre-determined proteins. The comparison is used to determine a corresponding absolute quantity of protein based on the observed sum or average of ionization intensities. A simple conversion factor can be applied to the calibration standard value to determine the absolute quantity of protein in the sample.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/914,578 filed Aug. 1 2008, (now U.S. Pat. No. 8,271,207, issued Sep.18, 2012), which is the National Stage of International Application No.PCT.US 2006/021517, filed Jun. 2, 2006, which claims the benefit of U.S.Provisional Application No. 60/686,967, filed Jun. 3, 2005, all of whichare hereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to LC/MS analysis of proteinmixtures. More specifically, the present invention relates to absolutequantitation of proteins by LC/MS analysis of enzymatically digestedproteins in simple or complex mixtures.

2. Background of the Invention

The study of proteins is crucial in a number fields includingunderstanding and combating disease through identification of proteins,discovering disease biomarkers, studying protein involvement in specificmetabolic pathways and identifying protein targets in drug discovery. Animportant technique that is often used in these studies is liquidchromatography combined with electrospray ionization mass spectrometry(ESI-LC/MS) to quantitate and identify peptides and proteins present insimple and complex mixtures.

One approach for quantifying peptides and proteins in simple and complexmixtures involves determining the corresponding relative abundancebetween two experimental conditions. During these experiments it isimportant to compare identical components between the two experiments inorder to accurately determine relative ratios of peptides to particularprotein(s). By doing so, multiple relative abundance values for eachpeptide to a given protein can be obtained to quantitativelycharacterize the differential expression of proteins between and amongdifferent physiological conditions.

Another approach to the quantitative study of proteins is to determinethe absolute concentration of the peptides and/or proteins that resultfrom enzymatic digestion of a given protein sample. In this approach,digestion of a protein sample using a protease such as trypsin producesmany smaller polypeptides, each having a specific primary amino acidsequence. It is known that a given mole quantity of protein produces thesame mole quantity for each tryptic peptide cleavage product if theproteolytic digest is allowed to proceed to completion. Thus,determining the mole quantity of tryptic peptide to a given proteinallows determination of the mole quantity of the originating protein inthe sample. Absolute quantitation of the protein can then beaccomplished by determining the absolute quantity of the peptides tothat protein(s) in the digest mixture.

Typically, absolute quantitation of proteins requires one or moreexternal reference peptides that are used to generate a calibrationresponse curve for specific polypeptides from a given protein (i.e.,synthetic tryptic polypeptide product). The absolute quantitation of thegiven protein is determined from the observed signal response for thespecific polypeptide in the sample relative to that generated in thecalibration curve. If the absolute quantitation of a number of differentproteins is to be determined, separate calibration curves are generatedfor each specific external reference peptide for each protein.

U.S. Patent Application No. 2004/0229283 to Gygi et al. (“Gygi”)describes a conventional technique for absolute quantitation of proteinsin complex mixtures that uses a synthesized derivative peptide as astandard. A derivative peptide is a peptide that is chemically identicalto a naturally occurring peptide of a given protein. The derivativepeptide is introduced to a complex mixture. The mixture is analyzedusing LC/MS resulting in ionization intensities for the derivativepeptide. This intensity signal response is compared with an intensitycalibration curve created using the introduced synthetic molecule todetermine the amount of the derivative protein in the mixture. Adisadvantage with using synthetic peptides is that extra steps arerequired to synthesize an authentic sample, and to later “spike” thesynthetic standard prior to being able to determine the absolutequantity of the protein itself.

Another technique for absolute quantitation of proteins employs anS35-methionine or other types of radio label, whose specific activity isknown. In this radio labeling techniques, an amino acid, such asS35-methionine, that is radio labeled is fed to a cell. As proteins aresynthesized, the proteins incorporate the S35-methionine instead ofmethionine. Based on the extent of incorporation of the radio label, theabsolute amount of the peptide or protein can be determined. Adisadvantage with using radio labels is that in some instances, such asstudies on humans or other organisms, radioactive feeding or doping isexpensive and may be deleterious to the subject and thereforeimpractical. Consequently, determining absolute quantitation of proteinsusing radio label techniques is limited to expendable biological systemssuch as microbes and plants.

Other protein quantitation techniques provide relative quantitation ofprotein amounts between two samples. Relative quantitation providesinformation as to how specific protein abundances change due to aperturbation (environment-induced, drug-induced, disease-induced). But,such relative quantitation techniques do not provide the absolutequantity of a particular protein present in a sample.

Consequently, a technique for determining the absolute quantity of aprotein in a sample that does not suffer from the disadvantages orrequirements of the prior art is required.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention provide absolute quantitation ofproteins from LC/MS data of simple or complex mixtures of chemically- orenzymatically-generated peptides without requiring synthesis of externalreference peptide(s) or the implementation of radio labeling methods.Embodiments of the present invention use a single calibration standardthat is applicable to the subsequent absolute quantitation of all otherproteins.

In an embodiment of the present invention, one or more predeterminedcalibration standard proteins are chemically or enzymatically degradedto their corresponding polypeptide cleavage products. The resultingpolypeptide products are analyzed by LC/MS. A calibration standard tableof the average signal response of the top N most efficiently ionizingpeptides associated with one or more predetermined calibration standardproteins is created as a function of known quantity (moles). During anactual experiment, proteins in a mixture are degraded chemically orenzymatically into their corresponding polypeptide cleavage products andthe resulting polypeptide products are analyzed by LC/MS. For eachprotein present, the top N most intense polypeptides are selected andtheir corresponding intensities are averaged. The average signalresponse value from the top N most efficiently ionizing polypeptides ofa given protein in the sample is compared to the average signal responsevalues annotated in the calibration standard table to determine theabsolute quantity of each protein present. A conversion factor orinterpolation technique can be used to determine the absolute quantityof a protein(s) present when the average signal response value is not inthe calibration standard table.

In an embodiment of the present invention, the calibration standardtable is generated using one or more predetermined proteins. Theproteins are chemically or enzymatically treated to produce a set ofcharacteristic polypeptide cleavage products. The polypeptide mixture(s)are analyzed by LC/MS to generate an inventory of the polypeptide massesand their corresponding signal responses. The LC/MS analysis isconducted with one or more known absolute quantities of the one or morecalibration standard protein(s). The signal responses of the top N mostefficiently ionizing polypeptides are selected from each calibrationprotein and the average signal response is incorporated into thecalibration table. Using more calibration standard proteins over whichto calculate peptide ionization sums or averages reduces statisticalerror.

In one embodiment, the present invention is a method for absolutequantitation of proteins in a sample. The method of the embodimentincludes digesting the sample to obtain peptides associated with theproteins in the sample and analyzing the digestion products using anLC/MS apparatus to obtain an inventory of corresponding peptide massesalong with their observed signal response for a particular protein.Further, the method of the embodiment includes determining the N mostefficiently ionizing peptides observed from the LC/MS analysis for theparticular protein and calculating the sum or average signal responsefor the N most efficiently ionizing polypeptides. In addition, themethod of the embodiment includes comparing the calculated sum oraverage signal response to a calibration standard and determining anabsolute quantity of the particular protein present in the sample basedon the comparison.

In another embodiment, the present invention is a system for absolutequantitation of proteins in a sample. The system includes a massspectrometer to generate an inventory of polypeptide masses along withtheir corresponding signal response to a particular protein in thesample and a computer. The computer includes a memory for storing acalibration standard table having entries for one or more proteins, eachentry having a quantity of protein and the average signal response forthe N most intense polypeptides to that protein. Further, the computerincludes software executing thereon for enabling the computer to obtainionization data from the mass spectrometer corresponding to peptides ofthe particular protein, analyze the obtained peptide ionization data,calculate one of the sum and average of the top N highest observedintensities, and determine an absolute quantity of the particularprotein present in the sample by comparing the calculated sum or averageto one or more entries in the calibration standard table.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar plot showing the signal responses of peptides fromvarying quantities of yeast enolase.

FIG. 2 is a bar plot showing the signal responses of peptides fromvarying quantities of yeast alcohol dehydrogenase.

FIG. 3 is a bar plot showing the signal responses of peptides fromvarying quantities of rabbit glycogen phosphorylase.

FIG. 4 is a bar plot showing the signal responses of peptides fromvarying quantities of bovine serum albumin.

FIG. 5 is a bar plot showing the signal responses of peptides fromvarying quantities of bovine hemoglobin (alpha chain).

FIG. 6 is a bar plot showing the signal responses of peptides fromvarying quantities of bovine hemoglobin (beta chain).

FIG. 7 is a plot of the average of the top 3 most intense peptide signalresponses yeast enolase for the quantities illustrated in FIG. 1.

FIG. 8 is a plot of the average of the top 3 most intense peptide signalresponses yeast alcohol dehydrogenase for the quantities illustrated inFIG. 2.

FIG. 9 is a plot of the average of the top 3 most intense peptide signalresponses rabbit glycogen phosphorylase for the quantities illustratedin FIG. 3.

FIG. 10 is a plot of the average of the top 3 most intense peptidesignal responses bovine serum albumin for the quantities illustrated inFIG. 4.

FIG. 11 is a plot of the average of the top 3 most intense peptidesignal responses bovine hemoglobin (alpha and beta chains) for thequantities illustrated in FIGS. 5 and 6.

FIG. 12 is a composite plot of the signal responses shown in FIGS. 7 to11.

FIG. 13 is a composite normalized plot of the signal responses shown inFIGS. 7 to 11.

FIG. 14 is a flow chart for a method for generating a calibrationstandard table according to an embodiment of the present invention.

FIG. 15 is a flow chart for a method for using a calibration standardtable generated according to an embodiment of the present invention.

FIG. 16 is a table showing the top 3 most intense peptide signalresponses each of the proteins in FIG. 1-6 for each amount of theprotein tested.

FIG. 17 is a table that tabulates the sum of the signal responses foreach of the proteins in the table of FIG. 16.

FIG. 18 is a table that tabulates the average of signal responses foreach of the proteins in the table of FIG. 16.

FIG. 19 is a schematic diagram of a system for absolute quantitationaccording to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Proteins are made of a linear sequence of amino acids that togetherproduce a large, single polypeptide. Typically, during proteinquantitation studies, the original protein molecules are chemically orenzymatically degraded into smaller cleavage peptides (e.g., trypticpeptides). For example, digestion using the enzyme trypsin breaksproteins into tryptic peptides by cutting the protein on the C-terminalside of the amino acids lysine and arginine.

Although the resulting peptides can be analyzed using a massspectrometer, in general, because different peptides have differentionization efficiencies the signal response of the constituent peptidesare not the same for any particular protein. That is, some peptides aremore receptive to protonation/ionization than others. However, for anygiven protein the signal response of the tryptic peptides can be orderedto exhibit a Gaussian distribution. As a result, the relative abundanceof a protein can be determined by comparing the signal responses ofpeptides within a particular protein.

The inventors of the present invention discovered that from a serialdilution of equimolar levels of unrelated proteins, the average responsefrom the N most efficiently ionizing peptides of a protein is similaracross all proteins, where N is an integer. Other than the number ofpolypeptides produced from the enzymatic digestion there appears to beno effect regarding the size of the originating protein, the averagesignal response of the top N ionizing peptides from each protein issimilar regardless of the intact proteins molecular weight to within+/−20%.

Using this knowledge, the inventors of the present invention havedeveloped a system and method for absolute quantitation of proteins in asample. According to embodiments of the present invention, the top Npeptide signal responses for a particular protein are averaged. Assumingan equimolar amount of the protein, the average should be the same (towithin some error) for all proteins. Consequently, the average can becompared to a pre-determined calibration standard average (thatcorresponds to an amount of peptide) to determine the absolute quantityof the protein of interest.

As an example of the foregoing, five common proteins were studied:hemoglobin from a cow (14,000 molecular weight); alcohol dehydrogenasefrom yeast (25,000 molecular weight); enolase from yeast (50,000molecular weight); serum albumin from a cow (70,000 molecular weight);and glycogen phosphorylase (97,000 molecular weight). These proteinswere analyzed at a level high enough to obtain substantially all of thepeptides to the proteins.

FIGS. 1-6 are bar plots of signal response (counts) for varyingconcentrations of each of the proteins analyzed. The x-axis of each ofFIGS. 1-6 corresponds to the peptides in the protein (represented bytheir amino acid sequence). The y-axis of each of FIGS. 1-6 is theobserved ionization signal response from the LC/MS analysis of thepeptide mixture. Specifically, these varying concentrations are 5 pmol,2 pmol, 1 pmol, 0.5 pmol, 0.25 pmol and 0.10 pmol. Specifically, FIG. 1is a bar plot of the characterized peptides from yeast enolase (eno)digested using trypsin; FIG. 2 is a bar plot of the characterizedpeptides from yeast alcohol dehydrogenase (adh) digested using trypsin;FIG. 3 is a bar plot of the characterized peptides from rabbit glycogenphosphorylase (gp) digested using trypsin; FIG. 4 is a bar plot of thecharacterized peptides from bovin serum albumin (bs) digested usingtrypsin; FIG. 5 is a bar plot of the characterized peptides from bovinehemoglobin (alpha chain) (ha) digested using trypsin; and FIG. 6 is abar plot of the characterized peptides from bovine hemoglobin (betachain) (hb) digested using trypsin.

As seen in FIGS. 1-6, the signal responses associated with peptides fora particular protein can be arranged to exhibit a Gaussian distribution.This distribution suggests that broad categories of ionizationefficiency can be established for the peptides of a particularprotein—those that ionize well, those that ionize moderately, and thosethat ionize poorly. The ionization efficiency is directly related to theobserved signal response. Ionization efficiency further appears to be afunction of the presence of certain amino acids or sequences of aminoacids. That is, certain amino acids or sequences of amino acids are moreprevalent in peptides that ionize well than those that ionize less well.

FIG. 16 is a table 1600 showing the top 3 (i.e., N=3 in this example)peptide signal responses for each of the proteins in FIG. 1-6 for eachconcentration of the protein tested. FIGS. 17 and 18 are tables 1700 and1800 that tabulate the sums and averages, respectively, for each of theproteins in table 1600. In Tables 1700 and 1800, the signal responsevalues of ha and hb are added together for the sum and average signalresponses because hemoglobin contains equal amounts of ha and hb. As canbe seen, although the proteins are from different organisms and havedifferent molecular weights, the average signal response of the top 3ionizing peptides are virtually identical.

FIGS. 7-11 are plots of the average signal response for the 3 mostefficiently ionizing peptides for each of the proteins of FIGS. 1-6 as afunction of the protein concentration. FIG. 12 is a composite plot ofFIGS. 7-11. FIG. 13 is a composite normalized plot of FIG. 7-11. It isobserved that the response is virtually linear. FIG. 13, moreover,demonstrates that the normalized responses are virtually identicalindependent of protein. Both of these characteristics support theconclusion that absolute quantity of a protein can be reliablydetermined using signal responses associated with the top N mostefficiently ionizing peptides for a chosen one or more calibrationstandard proteins.

Thus, tables 1700 and 1800 provide a characteristic mole response forany peptide. For example, albumin can be selected as a calibrationstandard. Using mass spectrometry analysis of another protein, forexample, enolase, the N (for example, 3) most efficiently ionizingpeptides can be identified. Then the amount of the enolase that ispresent can be determined by comparing the signal response of the N mostefficiently ionizing peptides to the albumin standard.

As another example, where N is 3, if the sum of the N highest ionizingpeptides is on the order of 900,000 thousand counts from the MSanalysis, then from table 1700, it is estimated that 5 picomole (pmol)of enolase is present. Similarly, if the average of the N mostefficiently ionizing peptides is on the order of 300,000 counts, thenfrom table 1800, the amount of enolase present is determined to be 5μmol. Had the number of counts for the N most efficiently ionizingpeptides of enolase been on the order of 100,000 counts (33,333 countsaverage), then the estimate from table 1700 or table 1800 for the amountof enolase present would have been 0.5 μmol. From tables 1700 and 1800,it can be seen that there are approximately 180,000 counts (60,000counts average)/pmol of a particular protein present based on the N mostefficiently ionizing peptides. In general, for a particular count, anestimate of the amount of protein present is given as follows byequation (1) if sums are used, and equation (2) if averages are used.

$\begin{matrix}{Q = {\frac{{Observed}\mspace{14mu}{Sum}}{{Sum}\mspace{14mu}{in}\mspace{14mu}{Table}\mspace{14mu} 1}*{Corresponding}\mspace{14mu}{concentration}\mspace{14mu}{in}\mspace{14mu}{Table}\mspace{14mu} 2}} & (1) \\{Q = {\frac{{Observed}\mspace{14mu}{Avg}}{{Avg}\mspace{14mu}{in}\mspace{14mu}{Table}\mspace{14mu} 1}*{Corresponding}\mspace{14mu}{concentration}\mspace{14mu}{in}\mspace{14mu}{Table}\mspace{14mu} 3}} & (2)\end{matrix}$

For example, if the average intensity signal responses of the N mostefficiently ionizing peptides for a particular protein resulted in anaverage count of 650,000 counts, then using Equation 2 and Table 1800,an estimate for the absolute quantity of the protein is given by

${\frac{\text{650,000}}{\text{324,800}}*5\mspace{14mu}{pmol}} = {10\mspace{14mu}{{pmol}.}}$the value 324,800 is the calibration standard average data valuecorresponding to 5 μmol from Table 1800. Likewise, using the 0.50 μmolconcentration results in

${\frac{\text{650,000}}{\text{31,800}}*0.5\mspace{14mu}{pmol}} = {10.2\mspace{14mu}{{pmol}.}}$Using a unimolar calibration standard value eliminates the need for themultiplication because the corresponding concentration is unity,i.e., 1. For example, using the 1.00 μmol value from Table 1800 yields

$\frac{\text{650,000}}{\text{62,800}} = {10.4\mspace{14mu}{{pmol}.}}$These values are within an acceptable error of one another.

Alternatively, well-known interpolation techniques (such as, forexample, straight line, quadratic, polynomial, cubic spline) can be usedto determine the molar concentration corresponding to a particularcount.

The accuracy of these estimates can be provided by statistical analysis.Well known statistical analyses can be performed to provide confidencelevels for the estimates. For example, tables 1700 and 1800 demonstratethat the coefficient of variation for the counts for any of theconcentrations is within 20 percent. This is an acceptable range.

FIG. 14 is a flow chart for a method for generating a calibrationstandard table such as Tables 1700 and 1800 according to an embodimentof the present invention. In step 1402, one or more calibration standardproteins to be used for a calibration standard are identified. In step1404, a known amount (in pmols) of each identified calibration standardprotein is decomposed to generate its constituent peptides. For example,such decomposition can be digestion using the enzyme trypsin. The knownamount can also be obtained using prepared digests such as the MassPREP™peptide digestion standards (available for proteins such as Phophrylaseb, Yeast Enolase, Bovine Hemoglobin, Yeast Alcohol Dehydrogenase andBovine Serum Albumin) available from Waters Corporation, Milford, Mass.In step 1406, mass spectroscopic analysis is performed on thedecomposition result. In step 1408, the N most efficiently ionizingpeptides are identified. In step 1410, the sum or average of the N mostefficiently ionizing peptides is determined. In step 1412, the sum oraverage is stored, for example, in a table such as table 1700 or 1800along with the corresponding amount of protein. This process can berepeated for varying concentrations of protein. In an embodiment of thepresent invention, the table also includes the sum or average over allof the proteins, that is, a composite sum or average, for each of theconcentrations in the table.

This process is repeated for a number of different amounts of theprotein to generate a calibration standard table such as Table 1700 or1800. Although only one protein need be used for the calibrationstandard, averaging values for a plurality of selected calibrationstandard proteins is desirable to improve the statistics of thetechnique. The calibration standard table can also have only the averagesum or average of averages corresponding to each calibration standardprotein, as well as optionally a covariance of the average sum oraverage of averages.

Any number of peptides can be used as the set of N most efficientlyionizing peptides. Using fewer than 3 however, may result ininsufficient statistics to be accurate. As described, averages or sumscan be used to generate the calibration standards tables. Any one ormore proteins can be used as the set of calibration standard proteins.Any one or more different amounts of protein can be used to generate thecalibration standards tables. Using more proteins, provides estimates onthe coefficient of variation to provide additional confidence insubsequent analysis.

Larger proteins having more peptides are likely to have more peptidesshowing higher ionizations as more peptides increase the likelihood ofhaving amino acid sequences that result in higher ionization efficiency.Smaller proteins, which produce fewer tryptic peptides, are less likelyto have many peptides with amino acid sequences indicative of highionization efficiency. Consequently, N is likely to be able to be sethigher when larger proteins are being analyzed.

Storage of table 1700 precludes the need for regenerating thecalibration standard table for each experiment. Moreover, thecalibration standard table can be published or otherwise made availablefor others to use. For example, the table can be published in a journalor distributed by disk to interested users. Further, the table can bepublished on an Internet website, wherein distribution can befacilitated by a version of the table or tables that can be downloadedfrom the website. Numerous other methods for distributing such acalibration standard table would be well-known to those having skill inthe art. Publication in this manner can be particularly advantageouswherein a particular user community agrees on one or more proteins to beused as the calibration standard.

The calibration standard table also acts to provide a calibration for aparticular instrument. That is, the calibration determines, for aspecific instrument, the number of counts observed per mole for a givenprotein. This value may vary from instrument to instrument. However,once this value is determined through calibration, it is applicable tothe absolute quantitation of all proteins generated from the particularinstruments(s).

FIG. 15 is a flow chart for a method for using a calibration standardtable generated according to an embodiment of the present invention. Instep 1502, a sample containing a protein or complex mixture of proteinsis decomposed into its constituent peptides and run through the LCMS tocollect the data. As above, the decomposition can be digestion using theenzyme Trypsin. In step 1504, mass spectroscopic analysis is performedon the decomposition result. In step 1506, proteins are identified. Instep 1508, the N most efficiently ionizing peptides are identified foreach protein identified in step 1506. In step 1510, the sum or average(depending on the calibration standard table used) of the N mostefficiently ionizing peptides is determined for each of the proteins. Instep 1512, the sum (or average) is compared to the calibration standardtable. If the sum (or average) is present, then the corresponding amountof protein is used as the number.

If the sum or average is not present, then the comparison includescalculating the absolute amount of protein based on the comparison. Suchcalculation can include applying the conversion described above inequations (1) or (2), or using other well-known interpolation techniques(including, for example, straight line, quadratic, polynomial, cubicspline) can be used to estimate the molar amount of the protein based onthe counts. Alternatively, if the calibration standard table includescount data corresponding to a single mole of protein, then thecalculation can be dividing the observed count by the count in thecalibration standard table corresponding to one mole to obtain anestimate of the molar amount of a particular protein present.

As with generating the standard table, any number of peptides can beused as the set of N most efficiently ionizing peptides. Using fewerthan 3 however, may result in insufficient statistics to be accurate.

In either the case of generating the table or using the table, ifaverages are used, then N can be different for different proteins. Thismay reduce coefficients of variation when using larger proteins, therebyincreasing confidence in absolute quantitation estimates.

FIG. 19 is a schematic diagram of a system for absolute quantitationaccording to an embodiment of the present invention. The system includesa liquid chromatograph 1952 coupled to a mass spectrometer 1901. Massspectrometer 1901 is coupled to a computer 1914. Liquid chromatograph1952 can also be coupled to computer 1914. In the exemplary embodimentof the present invention illustrated in FIG. 19, mass spectrometer 1901is a Q-TOF instrument, available from Waters Corp. (Milford, Mass.). Inthe exemplary embodiment of the present invention illustrated in FIG.19, liquid chromatograph 1952 is a nanoACQUITY™ UPLC System or WaterCapLC, available from Waters Corp. (Milford, Mass.).

In operation, a protein mixture is either chemically or enzymaticallydegraded into peptide components, thereby forming a peptide mixture1950. Peptide mixture 1950 is separated in LC 1952. The separationcomponents are introduced to mass spectrometer 1901. One method for suchintroduction is using electrospray ionization to produce an analytespray 1902.

Analyte spray 1902 is introduced to a quadrupole section 1903 of massspectrometer 1901. In the quadrupole section, quadrupole 1904 is tunedto select a particular ion for subsequent analysis in time-of-flightsection 1905 of mass spectrometer 1901. The selected ion is fragmentedin collision cell 1906. The fragments are introduced into time-of-flightsection 1905. In time-of-flight section 1905, a pusher 1908 pushes thefragments toward a reflectron 1910. Reflection 1910 reflects the ions toa detector 1912. Detector 1912 detects ion intensities and forwards themto a computer 1914 for subsequent analysis. Computer 1914 executessoftware to analyze the fragments in accordance with embodiments of thepresent invention described above. Computer 1914 can be any computer andcomputer apparatus that can be configured to implement the presentinvention as described herein. Computer 1914 also includes memory 1916for storing a calibration standard tables such as calibration standardtable 1700. Memory 1916 can be any memory, internal or external, thatcan store table 1700 including for example, RAM, ROM, PROM, EPROM,EEPROM, magnetic disk, optical disk, or CD-ROM. A screen or display 1918is coupled to computer 1914 for displaying information to a user. Akeyboard 1920 is also coupled to computer 1914 to allow a user to enterdata. Keyboard 1920 can also have a mouse or other pointing devicecoupled thereto to assist the user in operating computer 1914 in a wellknown manner. Computers such as computer 1914 and its memory 1916 aswell as computer peripherals such as display 1918, keyboard 1920 andpointing device 1922 are well known to those skilled in the art, andneed not be described further.

The present invention provides not only a system and method fordetermining absolute quantity of proteins in a sample, but also a methodfor validating the quantitation. For example, assume an absolutequantitation for the protein glycogen phosphorylase was obtained usingand embodiment of the present invention. Assume further that only 5 highionization efficiency peptides were observed. FIG. 3 illustrates thatglycogen phosphorylase should have 8 high ionization efficiencypeptides. Consequently, observation of only 5 such peptides indicates apossible error in the experiment.

The foregoing disclosure of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Many variations andmodifications of the embodiments described herein will be apparent toone of ordinary skill in the art in light of the above disclosure. Thescope of the invention is to be defined only by the claims appendedhereto, and by their equivalents.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention.

What is claimed is:
 1. A method for absolute quantitation of proteins ina sample, comprising: receiving peptide ionization intensities for aparticular protein in the sample, wherein said peptide ionizationintensities are obtained by performing processing including massspectrometry performed by a mass spectrometer; determining an N highestpeptide ionization intensities observed for the particular protein;calculating one of a sum intensity of the N highest peptide ionizationintensities observed and an average intensity of the N highest peptideionization intensities observed; comparing the calculated sum intensityor the average intensity to a calibration standard; and determining anabsolute quantity of the particular protein present in the sample basedon said comparing.
 2. The method of claim 1, wherein N is greater orequal to three.
 3. The method of claim 1, further comprising generatinga calibration standard.
 4. The method of claim 3, further comprising:(a) obtaining a known quantity of a predetermined protein in acalibration standard mixture; (b) processing the calibration standardmixture to obtain peptides associated with the protein; (c) analyzingthe peptides to determine peptide ionization intensities correspondingto the peptides; (d) selecting an N highest peptide intensities; (e)calculating one of the sum and the average of the selected N highestpeptide intensities; and (f) storing the calculated sum or average in atable for later use as a calibration standard.
 5. The method of claim 4,wherein N is greater or equal to three.
 6. The method of claim 4,comprising repeating step (a)-(f) for one or more additionalpredetermined proteins.
 7. The method of claim 4, comprising repeatingstep (a)-(f) for one or more known concentrations of the predeterminedprotein.
 8. The method of claim 1, further comprising identifying theparticular protein.
 9. The method of claim 1, further comprisingcalculating the amount of the particular protein using interpolation.10. The method of claim 1, further comprising converting the observedamount based on quantities in the calibration standard.
 11. Anon-transitory computer readable medium comprising code stored thereonfor absolute quantitation of proteins in a sample, the non-transitorycomputer readable medium comprising code which, when executed by aprocessor, performs a method comprising: creating a calibration standardtable having entries for one or more proteins, each entry having anamount of protein and one of a corresponding sum or average signalresponse for that protein; obtaining peptides corresponding to a proteinin the sample; analyzing the obtained peptides using a mass spectrometerto obtain ionization intensities corresponding to the peptides;calculating one of a sum intensity of N highest peptide ionizationintensities obtained in said analyzing and an average intensity of the Nhighest peptide ionization intensities obtained in said analyzing; anddetermining an absolute quantity of the particular protein present inthe sample by comparing the calculated sum intensity or the averageintensity to one or more entries in the calibration standard table. 12.The non-transitory computer readable medium of claim 11, wherein N isgreater or equal to three.
 13. The non-transitory computer readablemedium of claim 11, wherein creating the calibration standard tablefurther comprises: (a) obtaining a known quantity of a predeterminedprotein in a calibration standard mixture; (b) processing thecalibration standard mixture to obtain peptides associated with theprotein; (c) analyzing the peptides to determine ionization intensitiescorresponding to the peptides; (d) selecting an N highest ionizingintensities; (e) calculating one of the sum and the average of theselected N highest ionization intensities; and (f) storing thecalculated sum or average in the calibration standard table.
 14. Thenon-transitory computer readable medium of claim 13, wherein N isgreater or equal to three (3).
 15. The non-transitory computer readablemedium of claim 13, wherein the method further comprises repeating steps(a)-(f) for one or more additional predetermined proteins.
 16. Thenon-transitory computer readable medium of claim 11, wherein the methodfurther comprises distributing the calibration standard table.
 17. Thenon-transitory computer readable medium of claim 11, wherein the methodfurther comprises making the calibration standard table available fordistribution on an Internet website.
 18. A non-transitory computerreadable medium comprising code thereon for absolute quantitation ofproteins in a sample, the non-transitory computer readable mediumcomprising code which, when executed by a processor, performs a methodcomprising: receiving peptide ionization intensities for a particularprotein in the sample, wherein said peptide ionization intensities areobtained by performing processing including mass spectrometry;determining an N highest peptide ionization intensities observed for theparticular protein; calculating one of a sum intensity of the N highestpeptide ionization intensities observed and an average intensity of theN highest peptide ionization intensities observed; comparing thecalculated sum intensity or the average intensity to a calibrationstandard; and determining an absolute quantity of the particular proteinpresent in the sample based on said comparing.
 19. The non-transitorycomputer readable medium of claim 18, wherein N is greater or equal tothree.
 20. The non-transitory computer readable medium of claim 18,wherein the method further comprises: obtaining a known quantity of apredetermined protein in a calibration standard mixture; processing thecalibration standard mixture to obtain peptides associated with theprotein; analyzing the peptides to determine peptide ionizationintensities corresponding to the peptides; selecting an N highestpeptide intensities; calculating one of the sum and the average of theselected N highest peptide intensities; and storing the calculated sumor average in a table for later use as a calibration standard.